Using single cell RNAseq to unveil characteristics of CD133+ neural stem cells during development Project Summary Neural stem cells (NSCs), by definition, are self-renewing, multipotent cells that generate major cell types of the nervous system. Our current knowledge about NSC is still quite limited, insufficient to fulfill the goal of achieving good understanding of neurodevelopment or to engage endogenous NSCs to repair degenerated or injured central nervous system (CNS). Like other tissue specific stem cells, NSCs reside in highly complex cellular microenvironment and are in close contact with both stem cell niche and their downstream progeny cell types, which had made the characterization of the cellular and molecular signatures of NSCs extremely challenging. Recently developed single cell RNAseq technology pave the way to uncover molecular characteristics of any cell types including stem cells with precision. Particularly, the novel droplet-based technique allowed thousands of single cells to be sequenced in one tube yet still produce transcriptomes with single cell resolution. In this application, we propose to apply this scalable and low cost single cell RNAseq analysis developed by 10X Genomics to characterize CD133 positive ependymal cells lining the ventricular surface of the brain. The transciptome data of thousands of single CD133+ cells from cortex at different time points during development will be subjected to comprehensive data analyses, including Weighted Gene Coexpression Network Analysis (WGCNA), Pseudo-time analysis, principle component analysis (PCA) / t- Distributed Stochastic Neighbor Embedding (tSNE). We aim to characterize the molecular features unique to CD133+ cell subtypes in a temporally and spatially specific manner, through which we can obtain a better understanding of NSCs activities during development, as well as their potential interactions with the vasculature niche. Immunohistochemistry and/or in situ hybridization analyses will be performed to validate the sequencing result and provide physical locations of each of the specific cell types. Through lineage tracing studies, we will be able to map out lineage relationships of heterogeneous NSC populations. We believe this study will provide valuable information for building the atlas or roadmap of the origins of NSCs and how they evolve during development. Moreover, we aim to bridge this project into a RO1 grant where the function of some of the interesting core regulatory genes identified by this study will be further studied by gain- and loss-of function analyses and lineage tracing studies.